Project Description/Abstract Defining the molecular interactions between a virus and its host that regulate gene-specific transactivation has been essential to understanding DNA virus persistence and replication. The Kaposi?s sarcoma-associated Herpesivrus Rta protein is necessary and sufficient for the virus to emerge from latency and replicate (lytic reactivation). Rta interacts directly with the cellular protein called RBP-Jk, which is also required for lytic reacti- vation. RBP-Jk normally specifies the genes that will be activated by the cellular Notch signal transduction pathway by binding sequence specifically to DNA. In this fashion, RBP-Jk serves as a ?landing pad? for the activated Notch receptor (Notch intracellular domain (NICD1)). KSHV Rta stimulates DNA binding of RBP-Jk during viral reactivation, a mechanism that is fundamentally different from the canonical mechanism established for other RBP-Jk-activating proteins, namely NICD1 and Epstein-Barr Virus (EBV) EBNA-2. Indeed, NICD1 is unable to stimulate complete viral reactivation, supporting a promoter-specific mechanism for controlling its activity in KSHV infected cells. Recent data suggest that DNA binding of RBP-Jk is regulated both positively and negatively in response to KSHV reactivation signals. Modulation of DNA binding of RBP-Jk is a novel level of regulation of the Notch pathway that has been underappreciated in the literature. The overall goal of this application is to define the basic molecular mechanisms that regulate RBP-Jk DNA binding in KSHV infected cells, and determine the transcriptional reprogramming that supports viral reactivation. Our studies will explain the fundamental regulation of productive and non-productive virus reactivation as determined by promoter- specific transactivation. We will therefore address these Specific Aims: Aim 1. Determine if novel host proteins stimulate Jk binding to viral promoters during KSHV reactivation. Aim 2. Determine how specific MBP/Jk/DNA complexes program Rta and Notch-dependent reactivation. A series of biochemical and molecular biological approaches are proposed. Protein-DNA interactions represent the basis for many of the experiments, and will be evaluated in response to overexpression or functional deletion of cellular proteins (termed ?motif binding proteins?, or MBPs). Effects on viral reactivation will be quantitated using a novel, highly quantitative, KSHV reporter virus. A major part of the project involves using a novel version of Rta to detect and measure its direct targets by next generation sequencing. This proposal will shed light on how Notch target genes are specified for transactivation, and reveal new components of the Notch signal transduction pathway.